The goal of this research is to develop novel, more effective therapies for the treatment of tuberculosis, including multidrug resistant tuberculosis and related atypical mycobacterial infections. Isoniaizid is one of the most widely used chemotherapeutics for the treatment of tuberculosis and the molecular target of isoniazid has been thought to lie in the biosynthetic pathways involved in cell wall construction. Isoniaizid is a prodrug that requires activation by an endogenous mycobacterial enzyme, the catalase-peroxidase KatG, to a toxic metabolite which then reacts with its specific target with lethal consequences. This initial activation step is important because clinical isolates which become resistant to isoniazid do so primarily by losing KatG function. Since M. tuberculosis (MTB) lives within a macrophage phagolysosome and is exposed to high concentrations of toxic oxygen species KatG has been proposed to be essential for intracellular survival. We have shown that the loss of KatG function in isoniaizid-resistant clinical isolates is accompanied in all cases by a second mutational event which results in the compensatory upregulation of a thioredoxin-dependent alkylhydroperoxidase called AhpC. Identification of the molecular target of activated isoniaizid is an extremely important goal since knowledge of this target would allow a rational redesign of isoniazid with the potential for removing the requirement for activation by KatG. The target for isoniazid in the isoniazid-resistant saprophytic mycobacterial species M. smegmatis has been determined to be an enoyl ACP reductase called InhA. However, in the highly isoniazid-sensitive MTB, InhA does not appear to be directly involved in isoniazid resistance. By examining two-dimensional gel electrophoretic patterns of pulse-labelled MTB following treatment with MIC concentrations of isoniazid we have identified an 80kDa protein which was uniquely and specifically upregulated. N-terminal amino acid sequencing of this protein revealed a strong homology to a small (12kDa) acyl carrier protein (ACP). Induction of this protein in the presence of radiolabelled isoniazid results in incorporation of the radiolabel into the 80kDa form. Further purification and characterization of this protein complex demonstrated that this was a covalent termolecular complex of isoniazid, ACP, and a 50kDa ketoacyl ACP synthase (KAS). Strikingly, the gene for this KAS is located immediately downstream of the ACP. Thus, the target of isoniazid action is the enzymatic extension of a 26-carbon fatty acid to the corresponding 28-carbon 2-ketoacyl ACP by this KAS enzyme.